阅读说明：本技术 一种基于直线电子加速器建立棉花辐照诱变文库的方法 (Method for establishing cotton irradiation mutagenesis library based on linear electron accelerator ) 是由 马雄风 孙旭武 刘祉辛 张先亮 代帅 梅峥 任翔 藏新山 刘浩 于 2021-08-06 设计创作，主要内容包括：本发明涉及作物辐照诱变育种领域,主要涉及到一种基于直线电子加速器建立棉花辐照诱变文库的方法。所述方法包括步骤：选择常规品种或者具有优良性状的棉花品种的种子,采用直线电子加速器辐照已包装的棉花种子,辐照剂量为250Gy/s,将辐照诱变处理过的种子播种,进行多代自交获得突变体库。根据本发明的方法,通过使用辐照用直线电子加速器对棉花种子进行高能电子束辐照诱变,具有简单、高效、安全、易于推广的特点。(The invention relates to the field of crop irradiation mutation breeding, and mainly relates to a method for establishing a cotton irradiation mutation library based on a linear electron accelerator. The method comprises the following steps: selecting seeds of a conventional variety or a cotton variety with excellent properties, irradiating the packaged cotton seeds by adopting a linear electron accelerator, wherein the irradiation dose is 250Gy/s, sowing the seeds subjected to irradiation mutagenesis treatment, and performing multi-generation selfing to obtain a mutant library. According to the method, the cotton seeds are subjected to high-energy electron beam irradiation mutagenesis by using the linear electron accelerator for irradiation, and the method has the characteristics of simplicity, high efficiency, safety and easiness in popularization.)

1. A method for establishing a cotton irradiation mutagenesis library based on a linear electron accelerator is characterized by comprising the following steps:

selecting seeds of a conventional variety or a cotton variety with excellent properties, and packaging;

irradiating the packed cotton seeds by a linear electron accelerator with the irradiation dose of 250Gy/s, and sowing the seeds subjected to irradiation mutagenesis treatment to obtain M₀Seed generation;

seeding said M₀Generating seeds, and screening single plants with target characters to harvest mutant seeds;

and (4) carrying out multi-generation selfing on the mutagenized seeds to obtain a mutant library.

2. The method for building cotton irradiation mutagenesis library based on the linear electron accelerator as claimed in claim 1, characterized in that mutagenized seeds are subjected to 3 generations of selfing to obtain mutant library.

3. The method for building cotton irradiation mutagenesis library based on linear electron accelerator as claimed in claim 1, characterized in that the cotton seeds are irradiated for 1 second.

Technical Field

The invention relates to the field of crop irradiation mutation breeding, and mainly relates to a method for establishing a cotton irradiation mutation library based on a linear electron accelerator.

Background

China is the first producing big country of the world with cotton yield, and cotton planting zones are roughly distributed in areas such as Xinjiang, Henan, Jiangsu, Hubei, Shandong, Hebei, Anhui and the like. At present, the cotton planting area is gradually reduced, but the consumption is gradually increased. The quality of domestic cotton is not high, and high-quality cotton varieties which can meet the requirements of cotton textile enterprises are lacked. Although the sequencing of cotton genome was completed since 2015, which provides a key reference for genetic information for molecular breeding and creation of new varieties of cotton, in the actual research and breeding process, the cotton research materials which can be used are mainly cotton ecotypes collected from all over the world, and the research and breeding requirements of one gene and one mutant are difficult to meet. In addition, despite the collection of a large number of cotton ecotypes, the excellent traits that can currently be used for new variety creation are extremely limited, and more abundant trait types continue to be created for research and breeding. The method of genetic mutagenesis is an effective method for rapidly creating a mutant library and screening excellent characters, but at present, no efficient and safe method system exists in the creation of the cotton mutant.

The irradiation mutagenesis is to change the genetic material of an organism by using high-energy rays, so that different character variation types appear in 0 generation and later generation. In crop mutation breeding, 2 mutation modes are commonly used: physical mutagenesis and chemical mutagenesis. The physical mutagenesis is mainly radiation, such as irradiation mutagenesis by electron beam, laser, X-ray, gamma ray, cosmic ray, etc. Chemical mutagenesis is also a commonly used mutagenesis approach in breeding research, and commonly used chemical mutagens include alkylating agents, azide compounds, base analogs, nitroso compounds, and the like.

At present, physical mutagenesis is widely applied to cotton breeding. The physical mutagen used in cotton mutation breeding includes gamma ray, space mutation, ion beam implantation, electron beam, etc. The research in the last 30 th century shows that the mutation rate of each character is less than 1% although the X-ray can cause the gene mutation of cotton. Although the properties of cotton can be effectively changed by space breeding, the cost is high and the popularization is difficult. Although in recent years use has been made of⁶⁰Co-gamma rays breed many good cotton varieties with a mutagenesis rate of up to a few thousandths, but have many disadvantages compared with electron beams.⁶⁰The object of Co-gamma ray radiation treatment is cotton stamen, anthers need to be collected firstly, mature pollen after mutagenesis treatment is collected for pollination, mutant seeds can be obtained after the seeds are mature, the test operation is complex, and the cultivation period is long. Ion beam implantation and electron beam irradiation are also used for treating cotton stamens, and have similar problems and low mutagenesis rate. The heavy ion beam mutagenesis cotton mutant needs to be placed in a radiation disc, the close point end of the seed is bombarded upwards, the operation is time-consuming and labor-consuming, and the seed bombardment quantity is limited.

In addition, because the efficiency of the existing mutant obtaining method is not limited, a cotton mutant library is not constructed, if high-energy electron beams can be used for high-efficiency irradiation mutagenesis, the cotton mutant library is expected to be quickly constructed, the cotton germplasm breeding work with high yield, improved cotton fiber quality, disease resistance and insect resistance is promoted, the use amount of chemical fertilizers and pesticides is reduced, and the implementation of an environment-friendly policy is facilitated. The creation of genetic materials plays an important role in the development of bioscience, induces plants to generate variation, constructs a mutant library, screens target mutants from the mutant library, and can provide abundant basic materials for the research of plant gene functions.

At the present stage, no literature report about electron beam irradiation of cotton seeds for mutagenesis is found, and the existing cotton seed mutagenesis method is implemented by bombardment with heavy ion beams, so that the operation is complex and the efficiency needs to be improved.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the above deficiencies of the prior art and provide a method for rapidly creating a cotton mutagenesis library based on a linear electron accelerator.

At present, no report is available in the prior art on the adoption of linear electron beams for mutagenesis of crops including cotton, and the technical system is not specially used for radiation mutagenesis breeding but used for comprehensive irradiation processing of various articles such as food, medical and health products, modified materials and the like.

The method for establishing the cotton irradiation mutagenesis library based on the linear electron accelerator comprises the following steps:

selecting seeds of conventional varieties or cotton varieties with excellent characters applied in large area in current agricultural production, and packaging;

irradiating the packed cotton seeds by a linear electron accelerator with the irradiation dose of 250Gy/s, and sowing the seeds subjected to irradiation mutagenesis treatment to obtain M₀Seed generation;

seeding said M₀Generating seeds, and screening single plants with target characters to harvest mutant seeds;

and (4) carrying out multi-generation selfing on the mutagenized seeds to obtain a mutant library.

The method for establishing the cotton irradiation mutagenesis library based on the linear electron accelerator comprises the step of carrying out 3-generation selfing on mutagenized seeds to obtain a mutant library.

The method for establishing the cotton irradiation mutagenesis library based on the linear electron accelerator is characterized in that cotton seeds are irradiated for 1 second.

According to the method, the cotton seeds are subjected to high-energy electron beam irradiation mutagenesis by using the linear electron accelerator for irradiation, and the method has the characteristics of simplicity, high efficiency, safety and easiness in popularization. Batch cotton mutants can be obtained in a short time, and technical support is provided for enriching cotton mutant libraries in China. With the success of cotton genome sequencing, a great deal of cotton gene sequence information has been obtained. To further elucidate the sequence information of cotton genomes, creating mutants of nucleic acid sequence variations in specific regions is a necessary way to study gene function. The establishment of a cotton mutant library with saturated diversity is a necessary condition of cotton functional genomics.

Drawings

FIG. 1 shows mutant seedlings obtained after irradiation mutagenesis;

FIG. 2 shows a 3-year inbred cotton mutant of Jijiaye;

FIG. 3 shows dwarfing cotton mutants obtained by 3 years selfing;

FIG. 4 shows a yellowing cotton mutant obtained by 3 years selfing;

FIG. 5 shows the 3-year inbred tufted stemless cotton mutants.

Detailed Description

Example 1

1) Selecting a conventional variety or a cotton variety with excellent properties which is applied in a large area in the current agricultural production, and selecting medium cotton 619 to be improved in the embodiment;

2) selecting good seeds, sealing and packaging the good seeds by a carton, wherein the indexes of the good seeds comprise full seeds, proper size and weight and clean epidermis, and then adopting a 10MeV/20kW reverse wave type electron accelerator (A)IS1020) to irradiate the packed cotton seeds, wherein the machine type can realize continuous irradiation of 10-20 tons of articles and realize automatic turnover of the articles;

3) sowing seeds subjected to irradiation mutagenesis treatment according to the irradiation dose of 250Gy, and counting the emergence rate after sowing to obtain the optimal irradiation dose, wherein the irradiation rate of the optimal irradiation mutagenesis is 250 Gy/s;

4) performing irradiation mutagenesis by using the optimal irradiation dose to obtain M₀Seed generation;

5) seeding said M₀Generating seeds, screening individual plants with target properties including leaf shape, size and color to obtain mutant seeds,plant height, seed size, cotton fiber length and quality, as shown in figure 1, and mutant seedlings obtained after irradiation mutagenesis;

6) a mutant library with a sample capacity of 30 ten thousand plants was obtained by a continuous 3-generation selfing test, and a cotton mutant with acer palmatum, a dwarf cotton mutant, a yellow cotton mutant and a cotton mutant without main stem were obtained by selfing for 3 years as shown in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.

Example 2 mutagenesis dose and mutagenesis efficiency

According to the technical scheme of the invention, the linear electron beam is applied to mutation breeding of cotton seeds for the first time. Designing mutagenesis doses of 150Gy, 250Gy and 350Gy, and carrying out cell experiments after mutagenesis, wherein the mutagenesis efficiency is counted, the mutagenesis efficiency of low dose 150Gy is too low, and the lethality/yellowing rate of the seedlings of the first generation F0 of mutagenesis is less than 1 percent; the lethality caused by high dose 350Gy is higher, and the lethality/yellowing rate of the mutagenic primary F0 seedlings is more than 30%; the medium dose of 250Gy is the optimal mutagenesis dose, the lethality/yellowing rate of the seedlings in the first generation of mutagenesis F0 is 10-20%, in the process of planting 30 ten thousand cotton plants and continuously selfing for 3 years, the mutation efficiency of the mutant library is higher and accords with the expectation, the mutation phenotype is stable, and the large-scale phenotype observation in the third year proves that the mutant phenotype is stable and accords with the basic expectation of the cotton mutant library.

TABLE 1 mutation status of cotton seeds treated with different mutagenesis parameters